Curcumin amino-acid ester derivative as well as synthesis method and application thereof

Abstract

The invention belongs to the technical field of drug research, and particularly relates to a curcumin amino-acid ester derivative as well as a synthesis method and application thereof. The curcumin amino-acid ester derivative has a structural formula as shown in a formula I in the specification. Experiments prove that the curcumin amino-acid ester derivative realizes inhibition of tumor growth and induction of tumor apoptosis by regulating signal targets such as AKT / Foxo1 in HCC cells, so that the anti-HCC efficiency is improved; and # imgabs0 #.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical research, and particularly relates to a curcumin amino acid ester derivative, a synthesis method thereof, and an application thereof. Background Art

[0002] Curcumin is a diketone polyphenol natural product extracted from the rhizomes of plants such as turmeric. It has good safety and has been approved by the Codex Alimentarius Commission (CAC) as an important food additive. It is one of the natural pigments that can be used in food and was the earliest to be promulgated in the "Hygienic Standards for the Use of Food Additives" in China. The National Cancer Institute (NCI) of the United States has listed it as a third-generation cancer chemopreventive drug, making curcumin more promising for development and being recognized as a leading compound for anti-tumor drugs.

[0003] Curcumin has a wide range of pharmacological activities, especially anti-tumor activity. Existing studies have shown that curcumin can inhibit the PI3K / AKT cell signaling pathway in a concentration- and time-dependent manner, down-regulate the mRNA levels of proteins PI3K, AKT, and mTOR, reduce the expression of tumor suppressor genes NF-κB and PTEN, and promote apoptosis of various tumor cells. Curcumin can inhibit the ERK and p38 MAPK signaling pathways of HCC cells SMMC-7721, down-regulate the expression of Bcl-2 and survivin, and at the same time up-regulate the expression of Bax and caspase-3, thereby inhibiting the proliferation of HCC cells and promoting their apoptosis. Curcumin can promote the expression of caspase-3 by reducing the phosphorylation levels of the tumor suppressor genes forkhead box protein O1 (Foxo1) and AKT, overexpressing miR-9, thereby inhibiting the proliferation of tumor cells and promoting apoptosis. Curcumin also affects the cellular redox balance, increasing the production of intracellular reactive oxygen species (ROS). However, excessive ROS can affect the regulation of pro-apoptotic proteins by members of the Bcl-2 family, leading to cell apoptosis.

[0004] However, curcumin has defects such as poor stability and low bioavailability, which limit its clinical application. The main reasons for the low bioavailability of curcumin mainly include: ① The unstable β-diketone structure is one of the main reasons for the low bioavailability of curcumin. ② Due to the presence of two phenolic hydroxyl groups in the curcumin structure, curcumin is easily metabolized into curcumin glucuronide and curcumin sulfate after oral administration, and can be transferred to parts outside the intestine, and this metabolite is easily cleared, resulting in a very short half-life of curcumin, thus accelerating metabolism and reducing bioavailability. Therefore, it is necessary to modify the structure of curcumin. Summary of the Invention

[0005] In view of the deficiencies of poor curcumin stability and low bioavailability, the present invention designs and synthesizes curcumin amino acid ester derivatives with relatively high bioavailability based on molecular dynamics simulation and structure-activity relationship research, and verifies their in vitro and in vivo anti-hepatocellular carcinoma activities at the cellular and animal levels.

[0006] To achieve the above object, the specific technical solutions provided by the present invention are as follows: The present invention provides a curcumin amino acid ester derivative, and its structural formula is shown in Formula I: Among them, R is selected from a hydrogen atom, an alkyl group, -CH2CH2SCH3, a tolyl group or 2-carboxypyrrole.

[0007] As a preferred embodiment of the present invention, the structural formula of the curcumin amino acid ester derivative is any one of the following a~g: 。

[0008] In the second aspect of the present invention, a method for synthesizing the curcumin amino acid ester derivative is provided, including the following steps: Vanillin and acetone are subjected to a condensation reaction under the catalysis of a base to obtain monoketocurcumin; The monoketocurcumin is mixed with an amino acid compound having an amino protecting group for a nucleophilic substitution reaction, and the product is deprotected to obtain the curcumin amino acid ester derivative; The synthesis route is as follows: Among them, the reaction conditions indicated by A are: acetone, aqueous sodium hydroxide solution, ethanol, 40 h to 55 h; the reaction conditions indicated by B are: (BOC)2O (di-tert-butyl dicarbonate), triethylamine, dichloromethane, 0 °C to room temperature, 12 h; the reaction conditions indicated by C are: EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide), DMAP (4-dimethylaminopyridine), N2, dichloromethane, 8 h to 15 h; the reaction conditions indicated by D are: hydrochloric acid methanol, 0 °C to room temperature, dichloromethane, 12 h.

[0009] As a preferred embodiment of the present invention, the molar ratio of vanillin to acetone is 1 to 3:1, and the molar ratio of monoketocurcumin to the amino acid compound having an amino protecting group is 1:2 to 2.5.

[0010] In the third aspect of the present invention, there is provided an application of the curcumin amino acid ester derivatives in the preparation of anti-tumor drugs.

[0011] As a preferred embodiment of the present invention, the curcumin amino acid ester derivatives are used for the preparation of drugs for treating liver cancer.

[0012] As a preferred embodiment of the present invention, the drug uses the curcumin amino acid ester derivatives as the sole effective active ingredient.

[0013] As a preferred embodiment of the present invention, the drug is prepared by compounding the curcumin amino acid ester derivatives with excipients.

[0014] As a preferred embodiment of the present invention, the drug is prepared into an oral preparation or an injection preparation according to a pharmaceutically acceptable method. During specific preparation, appropriate excipients or carriers can be selected, and the types and amounts of the excipients or carriers are specifically selected according to the dosage form of the drug to be prepared, such as disintegrants, wetting agents, thickeners, tackifiers, suspending agents, stabilizers, pH regulators, antioxidants, colorants, flavoring agents, preservatives, etc.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: Previous studies of the present invention have shown that monocarbonyl curcumin cinnamyl ester and monocarbonyl curcumin phenyl methyl ester can significantly improve the activity against human hepatocellular carcinoma (HCC) by converting the diketone structure of curcumin into a monoketone and modifying its benzene hydroxyl group with natural organic acids. The results of molecular dynamics simulations and pharmacological experiments show that the cinnamic acid carbon chain introduced in the previous studies of the present invention is too long. Subsequently, the research team of the present invention introduced phenyl ester compounds to shorten the carbon chain and make its structure more stable. However, both monocarbonyl curcumin cinnamyl ester and monocarbonyl curcumin phenyl methyl ester are hydrophilic. There are various transport proteins on the cell membrane, and hydrophilic drugs can be transported like "hitchhiking" with the help of these channel proteins, thus entering the cell more easily. At the same time, when the cell exchanges substances with the external environment, hydrophilic drugs can smoothly pass through the hydrophilic region on the cell membrane along the concentration gradient or with the assistance of carrier proteins, which helps them exert their pharmacological effects in vivo. Therefore, the research of the present invention focuses on appropriately shortening the carbon chain modification of the hydroxyl group on the benzene and introducing a part with better water solubility.

[0016] Based on the previous activity results, and on the basis of the established structure-activity relationship, this invention focuses on the structural modification of the β-diketone structure and the exposed hydroxyl groups of curcumin, retains the monocarbonyl structure of curcumin, abandons the less active chalcone structure, shortens the carbon chain of the ether bond on the benzene ring, and designs and synthesizes a series of curcumin amino acid ester derivatives. Due to the introduction of amino acid ester compounds, these derivatives shorten the carbon chain while modifying the phenolic hydroxyl group, and the resulting derivatives are also hydrophilic, which is more conducive to their exerting pharmacological effects in the body, laying a foundation for the further development of highly efficient and safe curcumin anti-hepatocellular carcinoma drugs, and at the same time reducing the drug toxicity and side effects.

[0017] The curcumin derivatives provided by this invention inhibit tumor growth and induce tumor apoptosis by regulating signal targets such as AKT / Foxo1 in HCC cells, thereby improving the efficiency of anti-HCC, and providing a theoretical basis for obtaining highly efficient and low-toxic anti-HCC drugs. Brief Description of the Drawings

[0018] Figure 1 are the interaction mode diagrams of curcumin, compound g, monocarbonyl curcumin compound, and endogenous ligand with Foxo1 protein (6AK4) respectively; Figure 2 is the plasma concentration-time curve of curcumin and its derivative g in rats (x±s, n = 5); Figure 3 is the anti-proliferation ability of (A) 10 μM compounds a - g and (B) different concentrations of compound g (0.5, 1, 2, 4, 8, 16, 32, 64 μM) on HepG2 cells determined by MTT method; Figure 4 is the cell cloning situation after treating hepatocellular carcinoma cells with compound g for 24 h; Figure 5 is the colony formation rate of cells after treating hepatocellular carcinoma cells with compound g for 24 h; Figure 6 is the cell migration situation after treating hepatocellular carcinoma cells with compound g; A. Scratch test to observe cell migration and statistical data; B. Crystal violet staining to observe cell migration and statistical data; Figure 7 is the change in the content of cell cycle-related proteins p27 and cyclinB1 after treating with different concentrations of compound g determined by Western blot method; Figure 8It is the change of ROS after treatment with compound g (H7 - 2, 4, 8 μM) determined by H2DCFDA fluorescent probe (A); the change in the content of ROS - related proteins gp91 phox, Nrf2, and HO - 1 after treatment with different concentrations of compound g (H7 - 2, 4, 8 μM) determined by Western blot (B) and after treatment with compound g (4 μM) for different times (C); Figure 9 It is (A) the change curve of nude mouse body weight; (B) the tumor volume in nude mice after treatment with compound g. Detailed implementation manners

[0019] The present invention can be further described through the following examples. However, the scope of the present invention is not limited to the following examples. Those skilled in the art can understand that various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention. The present invention generally and / or specifically describes the materials and test methods used in the experiments. Although many materials and operation methods used to achieve the purpose of the present invention are well - known in the art, the present invention still describes them in as much detail as possible here.

[0020] Curcumin has defects such as poor stability and low bioavailability, which limits its clinical application. Based on this, the present invention provides a curcumin amino acid ester derivative, and its structural formula is shown in Formula I:

[0021] Among them, R is selected from a hydrogen atom, an alkyl group, -CH2CH2SCH3, a tolyl group, or 2 - carboxypyrrole.

[0022] The synthetic route of this curcumin amino acid ester derivative is as follows: .

[0023] Example 1 First, an aqueous sodium hydroxide solution (2 M) was dropped into an ethanol solution (10 mL) of vanillin (1.52 g, 10 mmol) and acetone (5 mmol). After an aldol condensation reaction for 48 hours, a pale - yellow intermediate, namely monoketocurcumin, was obtained; Glycine (150 mg, 20 mmol) was dissolved in a culture flask with 20 mL of water and 40 mL of acetone. Triethylamine (1.5 equivalents) and (Boc)2O (22 mmol) were added with stirring at a controlled temperature of 0 - 40 °C for 4 h to obtain an amino - acid compound with an amino - protecting group, labeled as Boc - amino acid; Mono-ketocurcumin (1 g, 3.1 mmol) and Boc amino acid (7.5 mmol) were added to CH2Cl2 (25 ml) in a round-bottom flask. The catalyst 4-dimethylaminopyridine (1.5 mmol) was added to the reaction mixture and stirred for 0.5 h. After that, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (7.5 mmol) was added and stirred under a nitrogen atmosphere until the reaction was completed to obtain Compound 1;

[0024] Compound 1 (2 mmol) was reacted with 15 mL of 2 mol / L hydrochloric acid methanol solution at 0 °C for 2 h, and the reaction process was monitored by thin-layer chromatography. After the reaction was completed, the product was filtered, dried, and purified by recrystallization from ethanol and petroleum ether. This process gave curcumin amino acid ester derivatives, labeled as Compound a, with a yield of 77%. ESI-MS m / z: 439.45 (M-H) - 。

[0025] Example 2 First, an aqueous sodium hydroxide solution (2 M) was dropped into an ethanol solution (10 mL) of vanillin (1.52 g, 10 mmol) and acetone (5 mmol), and an aldol condensation reaction was carried out for 48 h to obtain a pale yellow intermediate, namely mono-ketocurcumin; Alanine (178 mg, 20 mmol) was dissolved in a culture flask with 20 mL of water and 40 mL of acetone, and triethylamine (1.5 equivalents) and (Boc)2O (22 mmol) were added with stirring at a controlled temperature of 0 - 40 °C for 4 h to obtain an amino acid compound with an amino protecting group, labeled as Boc amino acid; Mono-ketocurcumin (1 g, 3.1 mmol) and Boc amino acid (7.5 mmol) were added to CH2Cl2 (25 ml) in a round-bottom flask. The catalyst 4-dimethylaminopyridine (1.5 mmol) was added to the reaction mixture and stirred for 0.5 h. After that, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (7.5 mmol) was added and stirred under a nitrogen atmosphere until the reaction was completed to obtain Compound 2.

[0026] Compound 2 (2 mmol) was reacted with 15 mL of 2 mol / L hydrochloric acid methanol solution at 0 °C for 2 h, and the reaction process was monitored by thin-layer chromatography. After the reaction was completed, the product was filtered, dried, and purified by recrystallization from ethanol and petroleum ether. This process gave curcumin amino acid ester derivatives, labeled as Compound b, with a yield of 73%. ESI-MS m / z: 467.51 (M-H) - 。

[0027] Example 3 First, an aqueous sodium hydroxide solution (2 M) was dropped into an ethanol solution (10 mL) of vanillin (1.52 g, 10 mmol) and acetone (5 mmol). After an aldol condensation reaction for 48 hours, a pale yellow intermediate, namely monoketocurcumin, was obtained; Valine (234 mg, 20 mmol) was dissolved in a culture flask with 20 mL of water and 40 mL of acetone. Triethylamine (1.5 equivalents) and (Boc)2O (22 mmol) were added with stirring at a controlled temperature of 0 - 40 °C for 4 h to obtain an amino acid compound with an amino protecting group, labeled as Boc amino acid; Monoketocurcumin (1 g, 3.1 mmol) and Boc amino acid (7.5 mmol) were added to CH2Cl2 (25 ml) and placed in a round - bottom flask. The catalyst 4 - dimethylaminopyridine (1.5 mmol) was added to the reaction mixture and stirred for 0.5 h. After that, 1 - ethyl - (3 - dimethylaminopropyl)carbodiimide (7.5 mmol) was added and stirred under a nitrogen atmosphere until the reaction was completed to obtain Compound 3.

[0028] Compound 3 (2 mmol) was reacted with 15 mL of 2 mol / L hydrochloric acid methanol solution at 0 °C for 3 h, and the reaction progress was monitored by thin - layer chromatography. After the reaction was completed, the product was filtered, dried, and purified by recrystallization from ethanol and petroleum ether. This process gave a curcumin amino acid ester derivative, labeled as Compound c, with a yield of 72%. ESI - MS m / z: 523.61 (M - H) - 。

[0029] Example 4 First, an aqueous sodium hydroxide solution (2 M) was dropped into an ethanol solution (10 mL) of vanillin (1.52 g, 10 mmol) and acetone (5 mmol). After an aldol condensation reaction for 48 hours, a pale yellow intermediate, namely monoketocurcumin, was obtained; Leucine (262 mg, 20 mmol) was dissolved in a culture flask with 20 mL of water and 40 mL of acetone. Triethylamine (1.5 equivalents) and (Boc)2O (22 mmol) were added with stirring at a controlled temperature of 0 - 40 °C for 4 h to obtain an amino acid compound with an amino protecting group, labeled as Boc amino acid; Mono-keto curcumin (1 g, 3.1 mmol) and Boc amino acid (7.5 mmol) were added to CH2Cl2 (25 ml) in a round-bottom flask. The catalyst 4-dimethylaminopyridine (1.5 mmol) was added to the reaction mixture and stirred for 0.5 h. After that, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (7.5 mmol) was added and stirred under a nitrogen atmosphere until the reaction was completed to obtain Compound 4.

[0030] Compound 4 (2 mmol) was reacted with 15 mL of 2 mol / L hydrochloric acid methanol solution at 0 °C for 3 hours, and the reaction process was monitored by thin-layer chromatography. After the reaction was completed, the product was filtered, dried, and purified by recrystallization from ethanol and petroleum ether. This process gave curcumin amino acid ester derivatives, labeled as Compound d, yield: 72%. ESI-MS m / Z: 551.67 (M-H) - 。

[0031] Example 5 First, an aqueous sodium hydroxide solution (2 M) was dropped into an ethanol solution (10 mL) of vanillin (1.52 g, 10 mmol) and acetone (5 mmol), and a pale yellow intermediate, namely mono-keto curcumin, was obtained through an aldol condensation reaction for 48 hours; Methionine (298 mg, 20 mmol) was dissolved in a culture flask with 20 mL of water and 40 mL of acetone, and triethylamine (1.5 equivalents) and (Boc)2O (22 mmol) were added with stirring at a controlled temperature of 0 - 40 °C for 4 h to obtain an amino acid compound with an amino protecting group, labeled as Boc amino acid; Mono-keto curcumin (1 g, 3.1 mmol) and Boc amino acid (7.5 mmol) were added to CH2Cl2 (25 ml) in a round-bottom flask. The catalyst 4-dimethylaminopyridine (1.5 mmol) was added to the reaction mixture and stirred for 0.5 h. After that, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (7.5 mmol) was added and stirred under a nitrogen atmosphere until the reaction was completed to obtain Compound 5.

[0032] Compound 5 (2 mmol) was reacted with 15 mL of 2 mol / L hydrochloric acid methanol solution at 0 °C for 3 hours, and the reaction process was monitored by thin-layer chromatography. After the reaction was completed, the product was filtered, dried, and purified by recrystallization from ethanol and petroleum ether. This process gave curcumin amino acid ester derivatives, labeled as Compound e, yield: 78%. ESI-MS m / Z: 587.73 (M-H) - 。

[0033] Example 6 First, an aqueous sodium hydroxide solution (2 M) was dropped into an ethanol solution (10 mL) of vanillin (1.52 g, 10 mmol) and acetone (5 mmol). After an aldol condensation reaction for 40 hours, a pale yellow intermediate, namely monoketocurcumin, was obtained. Phenylalanine (330 mg, 20 mmol) was dissolved in a culture flask with 20 mL of water and 40 mL of acetone. Triethylamine (1.5 equivalents) and (Boc)2O (22 mmol) were added with stirring at a controlled temperature of 0 - 40 °C for 4 h to obtain an amino acid compound with an amino protecting group, labeled as Boc - amino acid. Monoketocurcumin (1 g, 3.1 mmol) and Boc - amino acid (7.5 mmol) were added to CH2Cl2 (25 ml) and placed in a round - bottom flask. The catalyst 4 - dimethylaminopyridine (1.5 mmol) was added to the reaction mixture and stirred for 0.5 h. After that, 1 - ethyl - (3 - dimethylaminopropyl)carbodiimide (7.5 mmol) was added and stirred under a nitrogen atmosphere until the reaction was completed to obtain Compound 6.

[0034] Compound 6 (2 mmol) was reacted with 15 mL of 2 mol / L hydrochloric acid methanol solution at 0 °C for 2 h. The reaction progress was monitored by thin - layer chromatography. After the reaction was completed, the product was filtered, dried, and purified by recrystallization from ethanol and petroleum ether. This process gave a curcumin amino acid ester derivative, labeled as Compound f, with a yield of 75%. ESI - MS m / z: 619.70 (M - H) - .

[0035] Example 7 First, an aqueous sodium hydroxide solution (2 M) was dropped into an ethanol solution (10 mL) of vanillin (1.52 g, 10 mmol) and acetone (5 mmol). After an aldol condensation reaction for 55 hours, a pale yellow intermediate, namely monoketocurcumin, was obtained. Proline (232 mg, 20 mmol) was dissolved in a culture flask with 20 mL of water and 40 mL of acetone. Triethylamine (1.5 equivalents) and (Boc)2O (22 mmol) were added with stirring at a controlled temperature of 0 - 40 °C for 4 h.

[0036] Monoketocurcumin (1 g, 3.1 mmol) and Boc amino acid (7.5 mmol) were added to CH2Cl2 (25 ml) in a round-bottom flask. The catalyst 4-dimethylaminopyridine (1.5 mmol) was added to the reaction mixture and stirred for 0.5 h. After that, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (7.5 mmol) was added and stirred under a nitrogen atmosphere until the reaction was completed to obtain Compound 7.

[0037] Compound 7 (2 mmol) was reacted with 15 mL of 2 mol / L hydrochloric acid methanol solution at 0 °C for 2 - 3 hours, and the reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the product was filtered, dried, and purified by recrystallization with ethanol and petroleum ether. This process yielded curcumin amino ester derivatives, labeled as Compound g.

[0038] Experimental Example 1 Molecular docking and molecular dynamics simulation Molecular docking was performed using the Glide module of Schrodinger 2017 software. The Foxo1 protein (PDB ID: 6AK4) was prepared according to the ProteinPreparation Wizard process and the docking lattice was generated. Small molecules were prepared according to the LigPrep process, and standard mode (SP) docking was carried out using the OPLS-2005 force field.

[0039] The results showed (Table 1 and Figure 1 ) that the docking value of Compound g with the Foxo1 protein (6AK4) was the lowest, superior to the endogenous ligand, monoketocurcumin, and curcumin in the control group. The interaction mode indicated that the hydrogen bonds formed by Compound g with the key proteins ASP233, ARG96, VAL237, and the non-covalent interactions such as π-π stacking with HIS231 were beneficial to improving the anti-hepatocellular carcinoma ability.

[0040] Table 1 Standard mode (SP) docking values of curcumin, Compounds a - g, monoketocurcumin, and endogenous ligand with the Foxo1 protein (6AK4) Experimental Example 2 Bioavailability evaluation of curcumin amino ester derivatives Ten SD rats were randomly divided into two groups with an equal number of males and females. They were respectively intragastrically administered with 500 mg / kg of curcumin and its derivative g. Approximately 0.5 mL of blood was collected from the orbital cavity before intragastric administration and at 0.25, 0.5, 1, 1.5, 2, 3, 5, and 8 h after intragastric administration into anticoagulant Ep tubes, and plasma was prepared by centrifuging at 4000 r / min for 10 min. The content of curcumin in rat plasma was determined by UPLC-MS / MS. The blood concentration-time curve was plotted using Graphpad Prism 8.0, and the key pharmacokinetic parameters such as the peak time tmax, peak concentration Cmax, elimination half-life t1 / 2, and area under the curve AUC were calculated using Winnonlin. The relative bioavailability (F) was calculated according to the formula F = AUC (derivative g) / AUC (curcumin) × 100%.

[0041] The results showed that after intragastric administration of 500 mg / kg of curcumin to rats, the peak was reached at about 0.5 h; after intragastric administration of compound g, the peak was reached at about 1 h. AUC0-8 indicated that the bioavailability of compound g was increased by 7.6 times compared with curcumin, as shown in Figure 2 and Table 2.

[0042] Table 2 Comparison of main pharmacokinetic parameters of curcumin and its derivative g in rats (x ±s) Experimental Example 3 Antitumor cell proliferation, migration and pro-apoptotic effects of curcumin amino acid ester derivatives 0. Cell proliferation viability Hep G2 liver cancer cells in the logarithmic growth phase were seeded in 96-well plates and cultured in 1640 medium containing 10% FBS for 24 h. Cells were treated with different concentrations (0.5, 1, 2, 4, 8, 16, 32, 64 μM) of compound g, curcumin, and 10 μM of compounds a - g and curcumin for 24 h, 10 μL of MTT (5 mg / mL) was added to each well and the culture was continued at 37 °C for 4 h. Then, 100 μL of DMSO was added to each well and shaken at room temperature for 10 min, and the optical density was read at 490 nm on an enzyme-linked immunosorbent assay reader.

[0043] As Figure 3 shown in 50 A and B, compared with curcumin (IC 50 = 34.29 μM), derivative g had stronger inhibitory proliferation ability (IC

[0044] = 9.75 μM). Hepatocellular carcinoma cells Hep G2 in the logarithmic growth phase were seeded in 6-well plates at a density of 1000 cells / well. After overnight incubation, the cells were treated with the drug for 24 h. The medium was replaced with fresh medium, and the cells were further cultured at 37 °C until visible cell colonies formed. The cells were fixed with 4% paraformaldehyde and stained with crystal violet for 15 min.

[0045] The difference in the drug's colony formation ability was evaluated based on the number and size of the clones.

[0046] As Figure 4 、 5 shown, compared with the curcumin (IC 50 = 34.29 μM) group and the control group, the colony formation rate of derivative g decreased significantly, indicating that it had a significant effect on cell colony formation ability.

[0047] 2. Cell migration ability Hep G2 cells in the logarithmic growth phase were seeded in 6-well plates and incubated overnight until the cell confluence reached 100%. A scratch was made perpendicular to the bottom of the well plate. The cells were washed three times with 1×PBS and then cultured in serum-free medium. The cells were treated with curcumin (10 μM) and derivative g (10 μM) respectively. The cells were placed in an incubator at 37 °C and 5% CO2, and photographed at 0 h, 24 h, and 48 h to calculate the cell migration rate.

[0048] As Figure 6 shown in A, compared with the control group and the curcumin group, Hep G2 cells treated with derivative g migrated much slower in a time-dependent manner.

[0049] 3. Cell invasion ability Hep G2 cells in the logarithmic growth phase were used to prepare a 200 μL cell suspension in serum-free medium and seeded in the upper chamber of the transwell. The cells were treated with the above different drugs for 24 h. 500 μL of medium containing 10% FBS was added to the lower chamber. After culturing at 37 °C and 5% CO2 for 48 h, the non-migrated cells on the upper layer of the filter membrane were gently wiped off with a cotton swab. The filter membrane was fixed with methanol for 10 min, stained with crystal violet at room temperature for 15 min, and photographed under an upright optical microscope.

[0050] As Figure 6 shown in B, compared with the control group and the curcumin group, the number of cells that invaded to the other side of the chamber membrane was counted, and the number of cells in the derivative g group decreased significantly, indicating that it inhibited cell invasion.

[0051] 4. ROS detection Cells in the logarithmic growth phase, Hep G2, were inoculated in six-well plates and cultured overnight. The cells were then intervened with derivative g (2, 4, 8 μM) for 24 h. DCFH-DA was diluted to 10 μmol / L with serum-free culture medium at a ratio of 1:1000. The culture medium was aspirated, and 1 mL of the diluted DCFH-DA was added to the six-well plates. The plates were incubated in a cell culture incubator at 37 °C and 5% CO2 for 20 min, washed three times with serum-free culture medium, and then detected using a fluorescence microplate reader (excitation wavelength: 488 nm, emission wavelength: 525 nm).

[0052] As Figure 8 shown, the intracellular ROS level increased after treatment with derivative g, and the contents of related proteins gp91 phox, Nrf2, and HO-1 increased in a concentration- and time-dependent manner.

[0053] Experimental Example 4 Study on anti-xenograft hepatocellular carcinoma mouse model Animal experiments were carried out with the approval of the Medical Ethics and Animal Care Committee.

[0054] The experimental animals were female BALB / c mice, which were housed in an SPF facility. A suspension of hepatocellular carcinoma cells Hep G2 in the logarithmic growth phase was collected and subcutaneously injected into the right dorsal subcutaneous area of nude mice at a dose of 100 μL per mouse and 5×10 7 / mL. When the tumor volume reached about 100 mm 3 , the mice were randomly divided into three groups of six each. According to the treatment plan, curcumin and derivative g were intraperitoneally injected into the mice at a dose of 5 mg / kg. During the planned drug administration period, the tumor size and body weight were measured every 3 days.

[0055] The tumor volume was calculated according to the following formula: V = L × W 2 × 1 / 2; where: V, volume; L, tumor length; W, tumor width.

[0056] Inter-group comparisons were made based on indicators such as tumor volume and the survival period of the model mice to evaluate the different anti-cancer effects of curcumin and derivative g.

[0057] After the treatment, the mice were sacrificed, and important organs such as the heart and liver were isolated for HE or IHC staining. The toxic side effects and safety of derivative g were evaluated in combination with the changes in the body weight of the model mice during the treatment period or / and drug-related lethality.

[0058] As Figure 9 shown, after intraperitoneal injection of derivative g (5 mg / kg), the body weights of the animals in each treatment group remained relatively stable, indicating that compound g had low toxicity. From the comparison of tumor volumes, derivative g also had the potential to inhibit tumor growth in vivo.

[0059] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above-described exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be embraced within the present invention.

Claims

1. A curcumin amino acid ester derivative, characterized in that: Its structural formula is shown in Formula I: Wherein, R is selected from a hydrogen atom, an alkyl group, -CH2CH2SCH3, a tolyl group or 2-carboxypyrrole.

2. The curcumin amino acid ester derivative according to claim 1, characterized in that The structural formula of the curcumin amino acid ester derivative is shown in any one of a to g below: 。 3. A method for synthesizing the curcumin amino acid ester derivatives, characterized in that: The following steps are involved: Vanillin and acetone undergo condensation reaction under base catalysis to obtain monoketone curcumin; The monoketone curcumin is mixed with an amino acid compound having an amino protecting group to carry out a nucleophilic substitution reaction, and the amino protecting group is removed from the product to obtain the curcumin amino acid ester derivative.

4. The method for synthesizing curcumin amino acid ester derivatives according to claim 3, characterized in that: The molar ratio of the vanillin to the acetone is 1-3:1, and the molar ratio of the monoketone curcumin to the amino acid compound having an amino protecting group is 1:2-2.

5.

5. The method for synthesizing curcumin amino acid ester derivatives according to claim 4, characterized in that: The condensation reaction is carried out at 0°C for 40h to 55h; The nucleophilic substitution reaction is carried out under the action of a catalyst at 0° C. to 25° C. for 8 h to 15 h.

6. Use of the curcumin amino acid ester derivative according to claim 1 in the preparation of anti-tumor drugs.

7. The use according to claim 6, characterized in that: The curcumin amino acid ester derivative is used for preparing anti-liver cancer medicine.

8. The use according to claim 6, characterized in that: The medicine uses the curcumin amino acid ester derivative as the only effective active ingredient.

9. The use according to claim 6, characterized in that: The medicine is prepared by compounding the curcumin amino acid ester derivatives with auxiliary materials.

10. The use according to claim 6, characterized in that: The drug is prepared into an oral preparation or an injection preparation according to a pharmaceutically acceptable method.

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